1) Field of the Invention
This invention relates to a novel heat-resistant .beta.-galactosyltransferase, its production process and its use. More specifically, this invention is concerned with a novel .beta.-galactosyltransferase produced by a microorganism belonging to the family of Actinomycetaceae--such as a microorganism belonging to the genus Saccharopolyspora--and having high heat stability, its production process and its use.
2) Description of the Related Art
Modification of a saccharide or glycoside by glycosylation is known to make it possible to impart new physiological activities or physical properties to the first-mentioned saccharide or glycoside (hereinafter simply called a "saccharide"). For example, such modification is known to enhance sweetness, to reduce or eliminate bitterness, to increase the water solubility of glycosides having low solubility in water (illustrative examples are found among active ingredients of Chinese herbal remedies and like ingredients), and/or to improve in vivo stability and intestinal absorption.
A function imparted as a result of glycosylation and its degree vary depending on the type of the glycosyl donor and also the nature of the saccharide so modified. There have however been many reports in which preferable results are obtained for the abovementioned objects by modifying saccharides with galactosyl groups. Based on such reports, a variety of function oligosaccharides and function glycosides have been increasingly developed.
For example, among oligosaccharides or saccharide-modified glycosides represented by the following formula: EQU Gal-(Gal).sub.m -X
in which Gal means a galactosyl group, X denotes a saccharide or glycoside and m stands for an integer, oligosaccharides (galactooligosaccharides) in which X is a glucosyl group (Glu) and m is an integer of 0-4 are known as proliferation promoting substances for Bifidobacterium bifidum, a benign intestinal bacterium (Japanese Patent Application Laid-Open "Kokai" No. SHO 55-104885). In addition, they are found to have a wide range of utility as food materials for their excellent sweetness intensity and quality, low cariogenecity, low calorific nature, high processing stability, good moisture retaining property, high water-activity lowering ability, good colorability, etc.
Further, galactosylation of sucrose provides galactosylsucrose of the above formula in which X is sucrose and m is 0 (Japanese Patent Application Laid-Open "Kokai" No. SHO 64-85090) whereas galactosylation of lactulose yields galactosyllactulose of the above formula in which X is lactulose and m is 0 (Japanese Patent Application Laid-Open "Kokai" No. SHO 63-94987). These modified saccharides are also found to have new functions as galactooligosaccharide has. In addition, as to the sweet glycoside rubsoside, improvements to its sweetness intensity and quality have been achieved by galactosylation Argic. Biol. Chem. 53, 2923-2928 (1989)!.
As has been described above, the transgalactosylation reaction by .beta.-galactosidase is utilized to add one or more galactosyl groups or oligogalactosyl groups so that a galactosylated product can be produced.
This reaction has its basis on the fact that some .beta.-galactosidases catalyze the .beta.-D-transgalactosylation reaction to a saccharide (or a saccharide moiety of a glycoside) in the presence of .beta.-galactopyranoside at a high concentration.
The levels of the ability of enzymes to transfer a .beta.-galactosyl group vary widely depending on their sources. To make the reaction proceed efficiently, it has been necessary to use a .beta.-galactosidase having high transgalactosylation activity.
Exemplary conventional .beta.-galactosidases include the enzyme derived from the mold fungus, Aspergillus oryzae (Japanese Patent Publication "Kokoku" No. SHO 55-104885), and the enzymes derived from the bacteria, Bacillus circulans (Japanese Patent Publication "Kokoku" No. SHO 62-209780) and Streptococcus thermophilus Food Chem. 10, 195-204 (1983)!. Galactooligosaccharide is actually produced by causing these enzymes to act on lactose. Further, examples of yeast cells having .beta.-galactosidase activities include Lipomyces, Rhodotrula, Sirobasidium, Sterigumatomyces Journal of the Agricultural Chemical Society of Japan, 63(3), 629 (1989)!, Sporoboromyces (Japanese Patent Publication "Kokai" No. SHO 62-208293), Cryptococcus (Japanese Patent Publication "Kokai" Nos. SHO 60-251896, SHO 62-130695 and SHO 61-236790), and Kluyveromyces (Japanese Patent Publication "Kokai" No. SHO 61-271999). Production of galactooligosaccharide making use of these yeast cells is also attempted.
Generally, as a donor of .beta.-D-galactosyl groups, use of lactose is most advantageous from the industrial viewpoint. Lactose is contained abundantly in cow milk and is also produced as a dairy waste abundantly in a large volume outside Japan, so that its price is lowest as a raw material. Incidentally, based on the fact that the production of lactose-hydrolyzed milk making use of a .beta.-galactosidase has already been practiced Food Chemical, 7, 38-44 (1986)!, a production process of processed galactooligosaccharide-containing milk, said process making use of a .beta.-galactose having high transfer activity, has been reported recently (Japanese Patent Application Laid-Open "Kokai" No. HEI 1-168234).
In general, a glycosyl transfer reaction proceeds faster and more efficiency as the concentration of a galactosyl donor ("lactose" in the present specification) becomes higher. For this reason, it is desirous to makes the concentration of lactose higher in the reaction mixture. However, a lactose solution of high concentration has high viscosity and tends to permit easy precipitation of crystals at room temperature, leading to the problem that its handling is difficult during the production steps.
It has hence been required to raise the temperature of the reaction system (for example, to 60.degree. C. or higher) so that precipitation of lactose can be suppressed and the viscosity can be lowered. It is advantageous from the standpoint of cost to increase the amount of the reactant (lactose or the like) to be charged per unit volume by increasing its solubility. Further, a chemical reaction proceeds faster as the temperature becomes higher. It is accordingly possible to increase the velocity of the enzyme reaction and hence to shorten the reaction time by raising the temperature of the reaction system. In addition, a higher reaction temperature makes saprophytes difficult to grow. Furthermore, it is also expected that bacteriostatic action takes place by the high osmotic pressure of the resulting high-concentration saccharide solution, said pressure having been achieved by the high temperature, and contributes to the prevention of saprophytic contamination during the production steps.
Although the transgalactosylation reaction at high temperatures has many advantages as has been described, the enzyme is required to have high heat stability in order to conduct the reaction at such a high temperature. Moreover, to advantageously use the above reaction in the industry, it is required to immobilize the enzyme and to make the reaction steps automatic and continuous so that the addition product can be mass produced and its production cost can be lowered.
A .beta.-galactosidase can be stabilized by lactose of high concentration in general. Still higher heat resistance is however required to immobilize the enzyme and to use it repeatedly at high temperatures over a long period of time. The enzymes derived from the mold fungus, Aspergillus oryzae, the enzymes derived from the bacteria, Bacillus circulans and Streptococcus thermophilus, and yeast cells having .beta.-galactosidase activities such as Lipomyces, Rhodotrula, Sirobasidium, Sporoboromyces, Cryptococcus and Kluyveromyces are not sufficiently high in heat stability. Their repeated use at high temperatures is therefore not suitable.
On the other hand, as a .beta.-galactosidase having high heat stability and capable of withstanding repeated use at high temperatures, the enzyme of Paecilomyces varioti Appl. Microbiol. Biotechnol., 27, 383-388 (1988)! is known. The optimal reaction pH of this enzyme is however 3.5, so that it is unsuitable for production steps in which lactose contained in cow milk (pH: approx. 7) is utilized.